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. 2002 Sep;76(18):8979-88.
doi: 10.1128/jvi.76.18.8979-8988.2002.

Inhibition of beta interferon transcription by noncytopathogenic bovine viral diarrhea virus is through an interferon regulatory factor 3-dependent mechanism

Susan J Baigent  1 , Gang Zhang, Martin D Fray, Helen Flick-Smith, Stephen Goodbourn, John W McCauley
Affiliations

Inhibition of beta interferon transcription by noncytopathogenic bovine viral diarrhea virus is through an interferon regulatory factor 3-dependent mechanism

Susan J Baigent et al. J Virol. 2002 Sep.

Abstract

The induction and inhibition of the interferon (IFN) response and apoptosis by bovine viral diarrhea virus (BVDV) has been examined. Here we show that prior infection of cells by noncytopathogenic BVDV (ncp BVDV) fails to block transcriptional responses to alpha/beta IFN. In contrast, ncp BVDV-infected cells fail to produce IFN-alpha/beta or MxA in response to double-stranded RNA (dsRNA) or infection with a heterologous virus (Semliki Forest virus [SFV]). ncp BVDV preinfection is unable to block cp BVDV- or SFV-induced apoptosis. The effects of ncp BVDV infection on the transcription factors controlling the IFN-beta induction pathway have been analyzed. The transcription factor NF-kappa B was not activated following ncp BVDV infection, but ncp BVDV infection was not able to block the activation of NF-kappa B by either SFV or tumor necrosis factor alpha. Furthermore, ncp BVDV infection did not result in the activation of stress kinases (JNK1 and JNK2) or the phosphorylation of transcription factors ATF-2 and c-Jun; again, ncp BVDV infection was not able to block their activation by SFV. Interferon regulatory factor 3 (IRF-3) was shown to be translocated to the nuclei of infected cells in response to ncp BVDV, although DNA-binding of IRF-3 was not seen in nuclear extracts. In contrast, an IRF-3-DNA complex was observed in nuclear extracts from cells infected with SFV, but the appearance of this complex was blocked when cells were previously exposed to ncp BVDV. We conclude that the inhibition of IFN induction by this pestivirus involves a block to IRF-3 function, and we speculate that this may be a key characteristic for the survival of pestiviruses in nature.

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Figures

FIG. 1.
FIG. 1.
Enhancement of SFV plaque formation by ncp BVDV. CaTe cells were either mock infected or infected with ncp BVDV (1 to 2 PFU/cell) and subsequently infected with dilutions of SFV. Cultures were fixed and stained between 18 and 66 h, and the sizes of SFV-induced plaques were measured. The figure shows the average size ± standard error of the mean of 40 to 50 plaques under each experimental condition in two independent experiments.
FIG. 2.
FIG. 2.
ncp BVDV does not affect induction of apoptosis by SFV. CaTe cells were either infected with ncp BVDV (1 to 2 PFU/cell) or left uninfected and were subsequently infected with SFV at ∼10 PFU/cell. Cells were harvested in sample buffer at hourly intervals from 2 to 8 h postinfection and were subjected to immunoblotting using an anti-PARP monoclonal antibody (11). (a) CaTe cells; (b) CaTe cells previously infected with ncp BVDV. M, mock-infected cells.
FIG. 3.
FIG. 3.
ncp BVDV inhibition of virus induction of an IFN-stimulated gene product, MxA. (a) ncp BVDV inhibition of MxA induction by SFV in CaTe cells. Cells were either mock infected or infected with ncp BVDV (1 to 2 PFU/cell) for 48 h. ncp BVDV-infected and mock-infected cultures were subsequently infected with SFV (0.01 PFU/cell) and incubated for 18 h. Cells were harvested in sample buffer and subjected to immunoblot analysis with a rabbit antiserum against human MxA protein. (b through e) Time course of induction of MxA and expression of viral NS3 and NS2/3 by cp BVDV in ncp BVDV-infected CaTe cells. CaTe cells were either mock infected for 48 h (b and d) or infected with ncp BVDV (1 to 2 PFU/cell) for 48 h (c and e). Cells were then either mock treated or infected with cp BVDV (2 to 3 PFU/cell). At the times indicated, cells were harvested in sample buffer and subjected to immunoblotting using either an antiserum against human MxA (b and c) or a bovine antiserum against BVDV proteins (d and e). MxA (a and b) or its expected position (c) is indicated. NS2/3 and NS3 (d and e) are BVDV polypeptides.
FIG. 4.
FIG. 4.
Effect of ncp BVDV infection on induction of MxA by dsRNA or by IFN-α in CaTe cells. CaTe cells were either mock infected (a and c) or infected at a high MOI with ncp BVDV (b and d) for 48 h. Cells were then treated for 20 h with fresh medium containing either various concentrations of recombinant bovine IFN-α1 (a and b) or the synthetic dsRNA poly(I)·poly(C) (c and d) at increasing concentrations as described in Materials and Methods. Cells were harvested in sample buffer and subjected to polyacrylamide gel electrophoresis and immunoblotting using a rabbit antiserum against human MxA. Lanes marked + in panels a and b indicate treatment with 10 μg of poly(I)·poly(C)/ml. M, mock treated. MxA (a, b, and c) or its expected position (d) is shown.
FIG. 5.
FIG. 5.
ncp BVDV inhibition of IFN-β gene transcription. CaTe cells were first either mock infected or infected with ncp BVDV (1 to 2 PFU/cell) for 48 h and then either mock infected or infected with SFV (5 PFU/cell) for the times indicated, or treated with poly(I)·poly(C) (dsRNA) by addition to the medium at 100 μg/ml for 2 h. Total RNA was prepared from the cells and analyzed for the presence of IFN-β and γ-actin mRNAs by RNase protection. The actin transcript is indicated. Multiple IFN-β transcripts are detected due to the limited cross-homology to the bovine probe used and the existence of at least three bovine IFN-β genes.
FIG. 6.
FIG. 6.
ncp BVDV has no effect on the induction of NF-κB. (a) CaTe cells were either mock infected (M) or infected with either ncp BVDV or cp BVDV (1 to 2 PFU/cell) for the indicated times. (b) CaTe cells were either mock infected or infected with ncp BVDV (1 to 2 PFU/cell) for 48 h and then either mock infected or infected with SFV (5 PFU/cell) for the times indicated. (c) CaTe cells were either mock infected or infected with ncp BVDV (1 to 2 PFU/cell) for 48 h and then either mock infected or infected with cp BVDV (1 to 2 PFU/cell). (d) CaTe cells were either mock infected or infected with ncp BVDV (1 to 2 PFU/cell) for 48 h and then either mock treated or treated with human TNF-α (10 ng/ml) for 90 min. Nuclear extracts were prepared from each experiment and analyzed by EMSA using the PRD II probe from the human IFN-β gene (41). This probe is able to bind NF-κB as the p65 homodimer and p50/p65 heterodimer and can also bind the unrelated transcription factor RBP-Jκ (S. Goodbourn and K. Mellits, unpublished data). The mobilities of the DNA-protein complexes are indicated at the left of each panel.
FIG. 7.
FIG. 7.
Activation of transcription factors in response to virus infection. (a) Effect of ncp BVDV on phosphorylation of JNK in SFV-infected CaTe cells. CaTe cells were either mock treated (first and third lanes) or infected with ncp BVDV at a high MOI (second and fourth lanes) for 48 h. Cells were then either mock treated (first and second lanes) or infected with SFV (∼10 PFU/cell) (third and fourth lanes). At 5 h postinfection, cells were harvested in sample buffer, run on a 10% minigel, and subjected to immunoblotting using a rabbit antiserum against the phosphorylated (activated) forms of JNK1 (46K) and JNK2 (57K). (b) Effects of ncp BVDV on phosphorylation and nuclear localization of IFN-β enhanceosome transcription factors. CaTe cells were either mock infected for 48 h (i, v, and ix), infected with ncp BVDV at a high MOI for 48 h (ii, vi, and x), infected with SFV at a high MOI for 6 h (iii, vii, and xi), or infected with SFV at a high MOI for 6 h in cells preinfected with ncp BVDV for 48 h (iv and viii). At these times cells were fixed, permeabilized, and then stained using either a rabbit antiserum specific for the phosphorylated form of c-Jun (i, ii, iii, and iv), an antiserum specific for the phosphorylated form of ATF-2 (v, vi, vii, and viii), or an antibody detecting IRF-3 (ix, x, and xi). For c-Jun-P and ATF-2-P, staining was revealed using a goat anti-rabbit secondary antibody conjugated to FITC and was examined using conventional fluorescence microscopy. For IRF-3, staining was revealed using a goat anti-rabbit secondary antibody conjugated to the fluorochrome ALEXA-488 and was examined using confocal microscopy.
FIG. 7.
FIG. 7.
Activation of transcription factors in response to virus infection. (a) Effect of ncp BVDV on phosphorylation of JNK in SFV-infected CaTe cells. CaTe cells were either mock treated (first and third lanes) or infected with ncp BVDV at a high MOI (second and fourth lanes) for 48 h. Cells were then either mock treated (first and second lanes) or infected with SFV (∼10 PFU/cell) (third and fourth lanes). At 5 h postinfection, cells were harvested in sample buffer, run on a 10% minigel, and subjected to immunoblotting using a rabbit antiserum against the phosphorylated (activated) forms of JNK1 (46K) and JNK2 (57K). (b) Effects of ncp BVDV on phosphorylation and nuclear localization of IFN-β enhanceosome transcription factors. CaTe cells were either mock infected for 48 h (i, v, and ix), infected with ncp BVDV at a high MOI for 48 h (ii, vi, and x), infected with SFV at a high MOI for 6 h (iii, vii, and xi), or infected with SFV at a high MOI for 6 h in cells preinfected with ncp BVDV for 48 h (iv and viii). At these times cells were fixed, permeabilized, and then stained using either a rabbit antiserum specific for the phosphorylated form of c-Jun (i, ii, iii, and iv), an antiserum specific for the phosphorylated form of ATF-2 (v, vi, vii, and viii), or an antibody detecting IRF-3 (ix, x, and xi). For c-Jun-P and ATF-2-P, staining was revealed using a goat anti-rabbit secondary antibody conjugated to FITC and was examined using conventional fluorescence microscopy. For IRF-3, staining was revealed using a goat anti-rabbit secondary antibody conjugated to the fluorochrome ALEXA-488 and was examined using confocal microscopy.
FIG. 8.
FIG. 8.
Effect of ncp BVDV on assembly of SFV-induced transcription factor complexes on an ISRE probe. CaTe cells were first either mock infected or infected with ncp BVDV (1 to 2 PFU/cell) for 48 h and then either mock infected or infected with SFV (5 PFU/cell) for the times indicated. Nuclear extracts were prepared from each experiment and analyzed by EMSA using the ISRE probe from the human ISG15 gene (43). This probe is able to bind a number of members of the IRF family, as demonstrated by the nine rightmost panels, which show the supershift patterns of extracts from CaTe cells infected for 4 and 8 h with SFV incubated with antisera to individual IRFs as indicated. Pi, preimmune serum. The mobilities of complexes containing distinct IRFs are indicated to the left of the gel.
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